Trypanosoma cruzi is an obligate intracellular protozoan parasite. In mammalian hosts T. cruzi cycles between a trypomastigote stage which circulates in the blood and the amastigote stage which replicates in the cytoplasm of infected host cells (primarily muscle).
T. cruzi is the etiological agent of Chagas disease and is ranked as the most serious parasitic disease in the Americas, with an economic impact far outranking the combined effects of other parasitic diseases such as malaria, schistosomiasis, and leishmania (Dias et al., Mem. Inst. Oswaldo Cruz, 1999, 94:Suppl.1:103). Chagas Disease affects up to 20 million individuals primarily in the Americas where the insect vectors are present and where zoonotic transmission cycles guarantee a steady source of parasites. T. cruzi infection has its greatest human impact in areas of Latin America where housing conditions bring people, infected animals, and vector insects into close proximity. More than 90 million are at risk of infection in endemic areas, and roughly 50,000 children and adults die of chronic Chagas disease every year due to lack of effective treatments. Additionally, 2-5% of fetus carried by infected mothers in endemic areas are either aborted or born with congenital Chagas disease. Loss of revenue in terms of productivity lost due to sickness and medical costs have an overwhelming effect on economic growth of these countries.
Recently, increasing travel and immigration have brought T. cruzi infection into the spotlight globally, even in areas where transmission has previously been absent or very low. T. cruzi has spread beyond the borders of Latin America and has been detected in Europe, Asia, and the United States (Ferreira et al., J. Clin. Micro., 2001, 39:4390). In the U.S., 50-100 thousand serologically positive persons progressing to the chronic phase of Chagas disease are present, and the number of infected immigrants in developed countries is increasing. It is expected that, due to the exponential increase in emigration from Latin America, Chagas disease may become a serious health issue in North America and Europe in the next decade.
Congenital and transfusion/transplantation-related transmissions are thus becoming increasingly recognized as significant threats. As the number of infected individuals grows, transmission of T. cruzi to non-infected individuals through blood transfusion and organ transplants from the infected immigrant donors is emerging as a route for T. cruzi transmission in more developed nations (Umezawa et. al. J. Clin. Micro., 1999, 37:1554; Silveira et. al. Trends Parasitol., 2001, 17; Chagas disease after organ transplantation—United States, 2001; MMWR Morb Mortal Wkly Rep. 2002 Mar. 15; 51(10):210-2). Each year, 15 million units of blood are transfused and approximately 23,000 organ transplants are performed in the United States alone, and presently almost none of the blood supply is tested for T. cruzi. A few cases of infection by T. cruzi through organ donation have already been reported to United States Centers for Disease Control since 2001. It has therefore become apparent that the screening of blood and organ donors is necessary not only in Latin America but also in developed countries that receive immigrants from endemic areas.
Diagnosis of T. cruzi infection is challenging for a number of reasons. The initial infection is seldom detected except in cases where infective doses are high and acute symptoms very severe, as in localized outbreaks resulting from oral transmissions. Classical signs of inflammation at proposed sites of parasite entry (e.g. “Romahia's sign”) or clinical symptoms other than fever, are infrequently reported. As a result, diagnosis is very rarely sought early in the infection, when direct detection of parasites may be possible. In the vast majority of human cases, T. cruzi infection evolves undiagnosed into a well-controlled chronic infection wherein circulating parasites or their products are difficult to detect even with the use of amplification techniques. A “conclusive” diagnosis of T. cruzi infection is often reached only after multiple serological tests and in combination with epidemiological data and (occasionally) clinical symptoms. Further complicating matters, some researchers have reported positive PCR and clinical disease in patients with negative serology. Salomone et al. Emerg Infect Dis. 2003 December; 9(12):1558-62.
Unfortunately, multiple studies from geographically distinct areas and utilizing a wide range of tests and test formats have shown current diagnostics to be far from dependable (Pirard et al., 2005, Transfusion 45: 554-561; Salomone et al., 2003, Emerg Infect Dis 9: 1558-1562; Avila et al., 1993, J Clin Microbiol 31: 2421-2426; Castro et al., 2002, Parasitol Res 88: 894-900; Caballero et al., 2007, Clin Vaccine Immunol. 14:1045-1049; Silveira-Lacerda et al., 2004, Vox Sang 87: 204-207; Wincker et al., 1994, Am J Trop Med Hyg 51: 771-777; Gutierrez et al., 2004, Parasitology 129: 439-444; Marcon et al., 2002, Diagn Microbiol Infect Dis 43: 39-43; Picka et al., 2007, Braz J Infect Dis 11: 226-233; Zarate-Blades et al., 2007, Diagn Microbiol Infect Dis 57: 229-232). Many of the most widely employed serological tests, including one recently licensed by the United States Food and Drug Administration for use as a blood screening test in the U.S. (Tobler et al., 2007, Transfusion 47: 90-96), use crude or semi-purified parasite preparations, often derived from parasite stages present in insects but not in infected humans. The most widely accepted serological tests for T. cruzi infection utilize antigens from either whole to semi-purified parasite lysates from epimastigotes that react with anti-T. cruzi IgG antibodies. These tests show a degree of variability due to a lack of standardization of procedures and reagents between laboratories, and a number of inconclusive and false positive results occur due to cross-reactivity with antibodies developed against other parasites (Nakazawa et. al. Clin. Diag. Lab. Immunol., 2001, 8:1024).
Other tests have incorporated more defined parasite components, including multiple fusion proteins containing epitopes from various parasite proteins, which, individually have shown some promise as diagnostics (Caballero et al., 2007, Clin Vaccine Immunol. 14:1045-1049; da Silveira J F et al., 2001, Trends Parasitol 17: 286-291; Chang et al., 2006, Transfusion 46: 1737-1744). Unfortunately, in the absence of a true gold standard, the sensitivity of new tests is generally determined using sera that have been shown to be unequivocally positive on multiple other serologic tests, but rarely with sera that are borderline or equivocal on one or more tests, an approach that assures only that the test being evaluated is no worse, but not necessarily any more sensitive, than the existing tests.